Analysis of the T. cruzi proteome from four life cycle stages will be accomplished using multi-dimensional liquid chromatography (LC) tandem mass spectrometry (MS/MS). The information derived from this analysis will be used in at least two ways to achieve the goals of the Program Project. First, we will identify expressed genes from among the predicted ORFs derived in Project 1. Second, this analysis will help us identify the stage specific expression of genes ? allowing us to target vaccination depending on the parasite stage that would be most likely affected. Three methods will be used to improve our coverage of the proteome: 1) development of better computational tools to match spectra to proteins more efficiently; 2) use of improved mass spectrometer for the analysis; and 3) evaluation of other types of LC separation strategies. Lastly, we will analyze sub cellular compartments of T. cruzi, specifically the cell surface proteins and secreted proteins from amastigotes in order to identify proteins that may be particularly attractive targets for immune recognition. The full utility of the proteome analysis will be achieved with the imminent completion of the T. cruzi sequencing project and the annotation of the genome by computational means and by using information we will generate from the proteome. The Bioinformatics core of this Program Project will play a crucial role in this project.